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Vectorial transport of nucleoside analogs from the apical to the basolateral membrane
in double-transfected cells expressing the human concentrative nucleoside
transporter hCNT3 and the export pump ABCC4*
Maria Rius, Daniela Keller, Manuela Brom, Johanna Hummel-Eisenbeiss,
Frank Lyko, and Dietrich Keppler
Division of Tumor Biochemistry (M.R., Da.K., M.B., Di.K.), Division of Epigenetics (M.R., J.H-
E., F.L.), and Light Microscopy Facility (M.B.), German Cancer Research Center, Heidelberg,
Germany
DMD Fast Forward. Published on April 1, 2010 as doi:10.1124/dmd.110.032664
Copyright 2010 by the American Society for Pharmacology and Experimental Therapeutics.
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RUNNING TITLE PAGE
Running Title: Vectorial transport mediated by hCNT3 and ABCC4
Corresponding author:
Maria Rius
Division of Epigenetics
German Cancer Research Center
Im Neuenheimer Feld 580
D-69120 Heidelberg, Germany
Phone: +49-6221-423806
Fax: +49-6221-423802
E-Mail: [email protected]
Number of text pages: 19
Number of tables: 2
Number of figures: 5
Number of references: 39
Number of words in Abstract: 250
Number of words in Introduction: 772
Number of words in Discussion: 1181
Abbreviations: 5-AzaCyd, 5-azacytidine; 5-aza-2'-dCyd, 5-aza-2'-deoxycytidine; 5-F-5'-
dUrd, 5-fluoro-5'-deoxyuridine; ABCC, human ATP-binding cassette transporter, subfamily C;
hCNT, human concentrative nucleoside transporter; hENT, human equilibrative nucleoside
transporter; MRP, human multidrug resistance protein; NBTI, S-(4-nitrobenzyl)-6-thioinosine).
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Abstract
The identification of the transport proteins responsible for the uptake and the efflux of
nucleosides and their metabolites enables the characterization of their vectorial transport and
a better understanding of their absorption, distribution, and elimination. Human concentrative
nucleoside transporters (hCNTs/SLC28A) are known to mediate the transport of natural
nucleosides and some nucleoside analogs into cells in a sodium-dependent and
unidirectional manner. On the other hand, several human multidrug resistance proteins
(MRPs/ABCCs) cause resistance against nucleoside analogs and mediate transport of
phosphorylated nucleoside derivatives out of the cells in an ATP-dependent manner. For the
integrated analysis of uptake and efflux of these compounds, we established a double-
transfected MDCKII cell line stably expressing the human uptake transporter hCNT3 in the
apical membrane and the human efflux pump ABCC4 in the basolateral membrane. The
direction of transport was from the apical to the basolateral compartment, which is in line with
the unidirectional transport and the localization of both recombinant proteins in the MDCKII
cells. Recombinant hCNT3 mediated the transport of several known nucleoside substrates
and we identified 5-azacytidine (5-azaCyd) as a new substrate for hCNT3. It is of interest
that coexpression of both transporters was confirmed in pancreatic adenocarcinomas, which
represent an important clinical indication for the therapeutic use of nucleoside analogs. Our
results thus establish a novel cell system for studies on the vectorial transport of nucleosides
and their analogs from the apical to the basolateral compartment. The results contribute to a
better understanding of the cellular transport characteristics of nucleoside drugs.
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Introduction
Purine and pyrimidine nucleoside analogs are important drugs for the treatment of
many tumors and viral infections (King et al., 2006). The identification and characterization of
the transport proteins involved in the entry and the efflux of these substances and their
metabolites across the plasma membrane represents an important contribution to a better
understanding of their targeting to certain cells, their efficacy, and toxicity. In addition, down-
regulation of the uptake transporter or up-regulation of the efflux pump, or a combination of
both can be a cause for the cellular resistance to nucleoside analogs and for therapeutic
failure (Gottesman, 2002). Several proteins have been described as transporters for
nucleosides, nucleoside analogs, and their metabolites (for reviews see King et al., 2006;
Deeley et al., 2006). Uptake of nucleosides and nucleoside analogs across the plasma
membrane is mediated, among others, by the members of the human concentrative
(CNTs/SLC28A) and equilibrative (ENTs/SLC29A) nucleoside transporter families, including
hCNT1, hCNT2, and hCNT3, as well as hENT1 and hENT2 (Zhang et al., 2007). After
intracellular phosphorylation, several members of the ATP-binding cassette (ABC)
transporter superfamily including ABCC4, ABCC5, and ABCC11 have been identified to
mediate the efflux of these compounds from cells (Deeley et al., 2006). So far, the
nucleoside transport proteins were mostly studied in transfected mammalian cells or by the
use of Xenopus oocytes expressing only one recombinant transport protein (Schuetz et al.,
1999; Chen et al., 2001; Wijnholds et al., 2000; Pratt et al., 2005; Guo et al., 2003). These
cell systems lacked the potential to characterize the uptake transporters together with the
export pumps in a single cell system and, in particular, in a polarized cell line useful for
studies on vectorial transport.
Vectorial transport of endogenous and xenobiotic substances across epithelial cells
results from the uptake into the cells and the subsequent unidirectional efflux of these
substances which may be unaltered or metabolized (Keppler, 2005). The intracellular and
extracellular concentrations of substances and their half-life in the organism are controlled by
vectorial transport. In the past years, double-transfected polarized cells expressing
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combinations of a basolateral uptake transporter together with an apical ATP-dependent
efflux pump were introduced and have been widely used as in vitro cell systems to
characterize transport proteins and to examine their substrate specificity (Cui et al., 2001;
Sasaki et al., 2002; Kopplow et al., 2005; Mita et al., 2006). These cell systems expressing
recombinant transport proteins represent valuable tools in drug discovery and they reflect
hepatic and renal epithelial vectorial transport, whereby the majority of compounds are taken
up across the basolateral membrane and effluxed across the apical membrane. However,
the reverse situation, i.e., the vectorial transport from the apical to the basolateral membrane
in polarized cells expressing an apical uptake transporter and a basolateral ATP-dependent
export pump has not yet been studied, even though it resembles, for example, the uptake
from the luminal side of the intestine, followed by basolateral efflux into blood. There are
uptake transporters that are only expressed in the apical membrane of epithelial cells and
export pumps that are only expressed in the basolateral membrane, which further
underscores the significance of apical to basolateral vectorial transport. Thus, these cell
systems allow studying the substrate specificity and the transport properties of uptake
transporters and efflux pumps in a cell line that resemble the in vivo state of epithelial cells
but also of non-epithelial cells that express several transport proteins.
The aim of the present work was to establish a polarized cell system with defined
human uptake and export transport proteins for the nucleoside and nucleoside analog
transport. As a nucleoside uptake transporter for this cell model, we selected hCNT3 since it
transports purine and pyrimidine nucleosides and various therapeutic nucleoside analogs in
a concentrative manner and with a broad substrate specificity (Ritzel et al., 2001; Sarkar et
al., 2005; Errasti-Murugarren et al., 2007). In addition, hCNT3 seems to concentrate
nucleosides intracellularly more efficiently than hCNT1 or hCNT2, and is thus expected to
play an important role in the pharmacokinetics of nucleoside analogs (Mangravite et al.,
2003). As an export pump, we chose ABCC4, which is known to confer resistance to
nucleosides and nucleotide analogs and functions as an ATP-dependent organic anion
transporter with broad substrate specificity (Russel et al., 2008). At the present time, the
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contribution of ABCC4 to resistance against nucleoside analogs has been recognized
(Schuetz et al., 1999), but further studies are required to prove its contribution under clinical
conditions and in patient samples. Here we report our studies on vectorial transport in a
double-transfected cell line stably expressing hCNT3 in the apical membrane and ABCC4 in
the basolateral membrane of polarized MDCKII cells.
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Methods
Chemicals
[2-14C]Cytidine (1.9 MBq/mmol), [5-3H]uridine (0.6 TBq/mmol), and [6-3H]5-fluoro-5'-
deoxyuridine (5-F-5'-dUrd; 0.1 TBq/mmol) were purchased from Moravek Biochemicals
(Brea, CA). [6-14C]5-Azacytidine (5-azaCyd) has been synthesized as described previously
(Rius et al., 2009). Unlabeled cytidine, 5-azaCyd, 5-aza-2'-dCyd, and uridine were obtained
from Sigma (St. Louis, MO). 5-F-5'-dUrd was from Sequoia Research Products (Pangbourne,
United Kingdom) and S-(4-nitrobenzyl)-6-thioinosine (NBTI) was from Biomol International
(Plymouth Meeting, PA). All other chemicals were of analytical grade and purchased from
Sigma, Merck (Darmstadt, Germany), or AppliChem (Darmstadt, Germany).
Human tissue samples
Human tissues were obtained as described previously (Schaub et al., 1999; König et al.,
2005; Rius et al., 2005). Freshly removed tissue samples were cut in the operating room and
were snap-frozen in liquid nitrogen and maintained at -80°C until further analysis by
immunoblotting and immunofluorescence microscopy. Informed consent for tissue delivery
was obtained from patients before surgical intervention.
Antibodies
The peptide corresponding to the 24 amino proximal amino acids of the human CNT3
sequence (NTSGNNSIRSRAVQSREHTNTKQD; the underlined letters NTS indicate the
antibody name; NCBI accession number NP_071410) was synthesized and coupled to
keyhole limpet hemocyanin (Peptide Specialty Laboratories, Heidelberg, Germany). Guinea
pigs were immunized with these conjugates to raise the polyclonal NTS (anti-hCNT3)
antibody. The SNG antiserum was raised against the carboxy terminus of the human ABCC4
sequence (SNGQPSTLTIFETAL; the underlined letters SNG indicate the antibody name;
(Rius et al., 2003). The monoclonal anti-Na+/K+ ATPase anti-mouse antibody was obtained
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from Sigma. The horseradish peroxidase–conjugated goat anti-rabbit IgG was from Bio-Rad
(Munich, Germany) and goat anti-guinea pig IgG was from Jackson Immunoresearch
Laboratories (West Grove, PA). Alexa Fluor 488-conjugated goat anti-rabbit and anti-guinea
pig IgGs were from Molecular Probes (Eugene, OR) and Cy3-conjugated goat anti-mouse
IgG was from Jackson Immunoresearch Laboratories.
cDNAs encoding human ABCC4 and hCNT3 proteins
The cDNA encoding ABCC4 (NCBI accession number NM_005845) was cloned into the
expression vector pcDNA3.1(-) (Rius et al., 2003). The cDNA encoding hCNT3 was cloned
from human ileum using hCNT3-specific primers (forward: 5’–
AGCATGGAGCTGAGGAGTACAGC–3’; reverse: 5’–TCAAAATGTATTAGAGATCCC–3’)
based on the original sequence published by Ritzel et al. (2001; NCBI accession number
NM_022127) and subcloned into the vector pCR2.1-TOPO (Invitrogen, Groningen,
Netherlands). The complete 2.1-kb cDNA insert was excised from the pCR2.1-TOPO vector
and cloned into the HindIII and XhoI restriction sites of the mammalian expression vector
pcDNA3.1(+) (Invitrogen). The hCNT3 protein encoded by the cloned hCNT3 cDNA was
100 % identical to the reference sequence (NCBI accession number NP_071410).
Cell culture and stable expression in mammalian cells
Madin-Darby canine kidney cells strain II (MDCK) were cultured as described previously (Cui
et al., 1999). MDCK cells were transfected with the pcDNA3.1(+)-hCNT3 cDNA construct or
vector only using Metafectene transfection agent (Biontex, München, Germany) according to
the manufacturer’s instructions. Stable transfectants were selected using medium containing
G418 (0.5 mg/ml). Resistant clones were induced with 10 mM sodium butyrate for 24 h to
enhance the expression of the recombinant protein (Cui et al., 1999) and screened by
immunoblot analysis and immunofluorescence microscopy for hCNT3 expression. For
generation of the CNT3/ABCC4 double-transfected MDCK cells, the CNT3 single-transfected
cells were transfected with the pcDNA3.1(-)-ABCC4 cDNA construct using Metafectene
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transfection agent. Selection was carried out with hygromycin B (500 µg/ml). After induction
with 10 mM sodium butyrate, resistant clones were screened by immunoblot analysis and
immunofluorescence microscopy for ABCC4 expression.
Preparation of crude membrane fractions, immunoblot analysis, and deglycosylation
Crude membranes were prepared from transfected MDCK cells in the presence of proteinase
inhibitors and analyzed by immunoblotting as described (Rius et al., 2003). The NTS
antiserum was diluted 1:1000 in PBS containing 0.05 % Tween 20, and the polyclonal SNG
antiserum was diluted as described previously (Rius et al., 2003). The horseradish
peroxidase–conjugated goat anti-rabbit and anti-guinea pig antibodies were used at a dilution
of 1:20000. Deglycosylation was performed as described (Rius et al., 2003).
Preparation of tissue homogenates.
Tissue (0.1 to 0.5 mg) was homogenized during thawing by 10-fold dilution with incubation
buffer (250 mM sucrose and 10 mM Tris/HCl, pH 7.4) supplemented with proteinase
inhibitors (0.3 µM aprotinin, 1 µM leupeptin and 0.1 mM phenylmethylsulfonyl fluoride). The
resulting suspension was further homogenized with a Potter-Elvehjem homogenizer at 1,000
rpm at 2 strokes per minute for 30 strokes at 4°C. The homogenate was centrifuged at
1,200 x g for 10 minutes at 4°C and the resulting supernatant was centrifuged at 100,000 x g
for 30 minutes at 4°C. The resulting pellet was homogenized in 10mM Tris/HCl buffer, pH
7.4, supplemented with proteinase inhibitors. Aliquots were stored at -80°C.
Immunofluorescence microscopy of cultured cells and tissue samples
MDCK cells were grown on ThinCert membrane inserts (diameter, 6 mm; pore size, 0.4 µm;
pore density, 1 x 108/cm2; Greiner Bio-One, Frickenhausen, Germany) for 3 days at
confluence and induced with 10 mM sodium butyrate for 24 h to enhance the expression of
recombinant proteins. MDCK cells were fixed for 30 min with 2 % paraformaldehyde in PBS
and permeabilized for 30 min in 1 % Triton X-100 in PBS. Cryosections (5 µm) were
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prepared with a cryotome (Leica, Bensheim, Germany), air-dried for at least 2 h and fixed in
pre-cooled acetone (-20°C for 10 min). hCNT3 was detected by the antiserum NTS, ABCC4
was detected by the purified antiserum SNG, and the basolateral membrane of tissue cells
was stained by antibody against the Na+/K+ ATPase. To test the specificity of the NTS and
SNG antibodies, the antibodies were pre-incubated for 2 h at room temperature with 90 µM
(final concentration) of the synthetic antigenic NTS and SNG peptides, respectively, before
their application to the tissue sections. Nuclei were stained with propidium iodide or with
DAPI. The membrane inserts were mounted onto glass slides using 50 % glycerol in PBS.
Images were taken with a confocal laser scanning microscope (LSM510 Meta, Carl Zeiss,
Oberkochen or SP5, Leica, Wetzlar, Germany).
Cytotoxicity assay
The sensitivity of MDCK cells to 5-azaCyd and 5-aza-2'-dCyd was assessed with AlamarBlue
assays (Biosource, Camarillo, CA). MDCK cells were seeded (1 x 104 cells per well) in 96-
well plates and incubated for 24 h before exposure to graded concentrations between 10 nM
and 100 µM of each drug for 72 h. The IC50 value was defined as the drug concentration
required to reduce cell survival, as determined by the relative absorbance of reduced
AlamarBlue, to 50 %.
Uptake studies
MDCK cells were seeded in six-well plates at a density of 2 x 106 cells per well and cultured
for 24 h at confluence. Cells were induced with 10 mM sodium butyrate for additional 24 h.
Uptake studies examining the Na+-dependent uptake of nucleosides were performed using
Na+-containing buffer (100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM
Hepes, pH 7.5) and Na+-free buffer in which NaCl was replaced by equimolar choline
chloride. Cells were first washed three times with Na+-free buffer and then incubated with 1
ml of Na+-containing buffer containing the labeled substrate at the indicated concentration.
After incubation at 37°C, cells were washed three times with ice-cold Na+-free buffer. For
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determination of kinetic constants in MDCK-CNT3 cells, transport rates were measured at
five different substrate concentrations (0.075, 0.1, 0.15, 0.25, 0.5, and 1 mM) over a 20 sec
incubation time. Intracellular radioactivity was determined after lysing the cells with 0.2 %
sodium dodecyl sulfate by liquid scintillation counting. Km values were determined as the
substrate concentration at half-maximal velocity of transport under these conditions by use of
double-reciprocal plots and direct curve-fitting to the Michaelis-Menten equation. Kinetic
analysis was not performed in MDCK-Co cells because they lacked significant levels of Na+-
dependent uptake.
Transcellular transport
MDCK cells were grown on ThinCert membrane inserts (diameter, 24 mm; pore size, 0.4 µm;
pore density, 1 x 108/cm2; Greiner Bio-One, Frickenhausen, Germany) for 3 days at
confluence and induced with 10 mM sodium butyrate for 24 h. The cells were washed in
prewarmed (37°C) Na+-containing or Na+-free buffer with 5 mM glucose. The radiolabeled
substrate was dissolved in Na+/glucose-containing or Na+-free/glucose buffer and added
either to the apical or to the basolateral compartment. After incubation at 37°C and at the
time points indicated, radioactivity was measured in the opposite compartment. When NBTI
was used, it was added to the basolateral compartment at a concentration of 10 µM. Cells
were washed with ice-cold Na+- and glucose-containing or Na+-free but glucose-containing
buffer. Intracellular radioactivity was determined after lysing the cells with 0.2 % sodium
dodecyl sulfate. Measurements at several time points in cells expressing recombinant
hCNT3 indicated that the transcellular transport from the apical to the basolateral
compartment was linear at least within the first 15 min in the presence of sodium. The
paracellular leakage was determined by the use of [3H]inulin (Biotrend, Köln, Germany) and it
amounted to less than 2 % of the radioactivity added for all MDCK cell lines. For statistical
analysis, the Student’s t test was used.
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Results
Expression and localization of recombinant hCNT3 and ABCC4 in MDCK cells. The
protein expression of hCNT3 and ABCC4 in the stably transfected MDCK cells was verified
by immunoblot analyses (Figs. 1A and B). The hCNT3 protein was specifically detected by
the polyclonal NTS antibody as two dominant broad bands, characteristic for glycosylated
proteins, with apparent molecular masses of 90 and 65 kDa in crude membranes from
MDCK-CNT3 and MDCK-CNT3/ABCC4 cells (Fig. 1A). Deglycosylation of the CNT3 protein
by PNGaseF shifted the two bands to a single band with a molecular mass of about 60 kDa
(not shown). ABCC4 was strongly expressed at 170 kDa in the MDCK-CNT3/ABCC4 cells
(Fig. 1B). In the vector-transfected control cells, none of the two transport proteins was
detectable. Control experiments showed that the NTS antiserum did not cross-react with the
recombinant hCNT1 expressed in MDCK cells (not shown).
The cellular localization of the recombinant transport proteins in the MDCK
transfectants was studied by confocal laser scanning microscopy (Figs. 1C-N). The NTS
antiserum localized hCNT3 in stably transfected MDCK cells (Figs. 1C-H) and optical vertical
sections showed intense red fluorescence for hCNT3 in the apical membrane domain of the
cells expressing CNT3 and CNT3/ABCC4 (Fig. 1D and F). No plasma membrane staining
was observed in vector-transfected MDCK-Co cells (Fig. 1G and H). ABCC4 was localized
exclusively to the basolateral membrane domain of the CNT3/ABCC4 double-transfected
MDCK cells (Figs. 1K and L). Our localization studies are in line with previous work that
showed apical localization of hCNT3 in MDCK cells (Errasti-Murugarren et al., 2007), and
basolateral localization of ABCC4 in MDCK cells expressing recombinant ABCC4 (Lai and
Tan, 2002).
Transport of [14C]5-azaCyd and other nucleosides mediated by hCNT3. To test the
functionality of hCNT3 in transfected MDCK cells, intracellular accumulation of several
nucleosides was measured in MDCK-Co and MDCK-CNT3 cells. The results showed that the
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MDCK-CNT3 transfectants mediated the Na+-dependent uptake of the known substrates
cytidine, uridine, and 5-FdUrd (Fig. 2B-D) with Km values of 151, 51, and 615 µM,
respectively (Table 1). Low transport rates of uridine were observed in the presence of
choline in MDCK-CNT3 cells (Fig. 2C) which accounted only for 20% of the Na+-dependent
uptake and were possibly due to transport by endogenous transporters. Uptake of 5-azaCyd,
a novel substrate identified recently for hCNT1 (Rius et al., 2009), was also analyzed in the
cells expressing hCNT3 (Fig. 2A). The Na+-dependent intracellular accumulation of
[14C]5-azaCyd at a concentration of 1 µM amounted to 221 pmol x mg protein-1 after 1 min in
MDCK-CNT3 cells (Fig. 2A). In contrast, the MDCK-Co cells did not mediate Na+-dependent
uptake of 5-azaCyd (Fig. 2A). Kinetic analysis showed a Km value of 147 µM (Table 1).
These results indicate that, in addition to established substrates, hCNT3 also mediates
cellular uptake of the anticancer drug 5-azaCyd.
hCNT3 expression enhances inhibition of the cell growth by 5-azaCyd and 5-aza-2'-
dCyd. The uptake and action of 5-azaCyd by hCNT3 was further studied in viability assays
with nonlabeled 5-azaCyd and with the structurally related derivative 5-aza-2'-dCyd. MDCK-
Co and MDCK-CNT3 cells were incubated with various concentrations of 5-azaCyd or 5-aza-
2'-dCyd for 72 h and cell viability was determined by AlamarBlue assay. The viability of
hCNT3-expressing MDCK cells decreased in a concentration-dependent manner in the
presence of 5-azaCyd or 5-aza-2'-dCyd (Table 2). The IC50 values obtained for MDCK-Co
and MDCK-CNT3 cells after incubation with 5-azaCyd were 54 ± 5 µM and 0.8 ± 0.07 µM,
respectively. Thus, hCNT3-expressing MDCK cells increased their relative sensitivity 68-fold
compared to MDCK-Co cells. After incubation with 5-aza-2'-dCyd, MDCK-Co, and MDCK-
CNT3 showed IC50 values of 331 ± 48 µM and 25 ± 4 µM, respectively, and the relative
sensitivity of MDCK-CNT3 cells thus increased 13-fold compared to MDCK-Co cells (Table
2).
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Vectorial transport of [14C]cytidine and [14C]5-azaCyd in MDCK cell expressing hCNT3.
MDCK cells were grown in a polarized fashion on cell culture inserts for studies on vectorial
transport. Figure 3A shows schematically the role of the recombinant hCNT3 in the vectorial
transport by the transfected MDCK cells. When MDCK cells were grown on filter membranes,
the recombinant hCNT3 mediated the uptake of nucleosides from the apical compartment
into the cells (Fig. 3A). To test whether vectorial transport of nucleosides takes place in the
correct direction, transcellular transport of nucleosides was measured across the apical and
the basolateral membrane and in the opposite direction in MDCK-CNT3 and MDCK-Co cells
(Fig. 3B). Incubation with [14C]cytidine at a concentration of 1 µM in the apical compartment
(apical→basol.) was associated with a Na+-dependent accumulation of radioactivity in the
basolateral compartment of MDCK-CNT3 cells (Fig. 3B, left panel). In contrast, when
radioactivity was added in the basolateral compartment (basol.→apical), accumulation in the
apical compartment was negligible (Fig. 3B, right panel). The Na+-dependent apical-to-
basolateral transport was 132-fold higher than that in the opposite direction in MDCK-CNT3
cells. Since we observed a substantial accumulation in the basolateral compartment (Fig. 3B,
left panel) without the expression of a recombinant efflux transporter in the basolateral
membrane, we raised the question whether basolateral transporters for nucleosides were
responsible for the release into the basolateral compartment and, in particular, whether the
canine equilibrative nucleoside transporters (Ents) may play a role in the transport across the
basolateral membrane of MDCK cells. ENTs are expressed in many cell types and function
as bidirectional nucleoside transporters that equilibrate intracellular and extracellular
concentrations of nucleosides (Zhang et al., 2007). To identify a potential role of canine Ents
in the observed basolateral efflux, we used NBTI to inhibit Ent-mediated transport in MDCK-
CNT3 cells. When transcellular transport across the apical to the basolateral membrane was
measured in the presence of 10 µM NBTI in the basolateral compartment, transcellular
transport in MDCK cells expressing hCNT3 became strongly reduced (Fig. 3C), indicating
activity of endogenous canine Ents in MDCK-CNT3 cells. In agreement with our previous
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findings, similar results were also obtained for the vectorial transport of 1 µM [14C]5-azaCyd
(data not shown).
Transcellular transport of [14C]cytidine and [14C]5-azaCyd mediated by CNT3/ABCC4
double-transfected MDCK cells. Finally, we sought to demonstrate that when the double-
transfected MDCK cells were grown on filter membranes, the recombinant hCNT3 mediates
the uptake of nucleosides from the apical to the intracellular space and the recombinant
ABCC4 mediates efflux of compounds from the intracellular space to the basolateral
compartment (Fig. 4A). The function of hCNT3 and ABCC4 in the double-transfected MDCK
cells was studied by measurement of the transcellular transport of the nucleoside analog
[14C]5-azaCyd (Fig. 4B-D), which is a substrate for hCNT3 (Fig. 2A). Thus, polarized MDCK
cells grown on ThinCert membrane inserts were incubated with [14C]5-azaCyd at
concentrations of 1 and 10 µM in the apical chamber compartment. After 15 min incubation,
the radioactivity accumulated in the cells (upper panels) and in the basolateral compartment
(lower panels) was measured. Figure 4B and 4C (upper panels) show the intracellular
accumulation of 5-azaCyd (1 µM) obtained in the presence of Na+-containing or of Na+-free
(choline) buffer. The Na+-dependent intracellular accumulation of 5-azaCyd was much higher
in MDCK cells expressing hCNT3 and ABCC4 than in the MDCK-Co cells (Fig. 4B-C, upper
panel), indicating that the apical hCNT3 transporter was responsible for the unidirectional
Na+-dependent uptake and intracellular accumulation of 5-azaCyd. The double-transfected
MDCK-CNT3/ABCC4 cells showed a similar Na+-dependent intracellular accumulation of
5-azaCyd as the single-transfected MDCK-CNT3 cells (Fig. 4B-C, upper panel). However the
transcellular transport of 1 µM 5-azaCyd did not differ significantly between the both cell lines
expressing hCNT3.
Because our previous results had suggested that the expression of endogenous
canine Ents may influence the transcellular transport in MDCK cells, we tested the effect of
NBTI in the transcellular transport of 5-azaCyd (Fig. 4C). When NBTI (10 µM) was added to
the basolateral compartment, transcellular transport was significantly detected only in the
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presence of Na+ and only in the MDCK cells expressing hCNT3 and ABCC4 (Fig. 4C, lower
panel). In addition, we also measured transcellular transport of 5-azaCyd at a higher
concentration of 10 µM and without NBTI (Fig. 4D). Under these conditions and in the
presence of Na+-containing buffer, we obtained a 2-fold higher transcellular transport in the
double-transfected cells expressing hCNT3 and ABCC4 than in the single-transfected cells
expressing only hCNT3 (Fig. 4D, lower panel, closed bars, p < 0.05). Because small
amounts of 5-azaCyd also entered the cells in the presence of Na+-free (choline) buffer, we
also observed a low level of transcellular transport in the absence of Na+ (Fig. 4D, lower
panel, open bars). This transcellular transport was approximately 3-fold higher in the MDCK-
CNT3/ABCC4 compared to the MDCK-CNT3 cells (Fig. 4D, lower panel, open bars).
ABCC4-mediated transcellular transport of 5-azaCyd at higher concentrations can be
explained by the higher saturation of canine Ents, thus allowing for a higher rate of transport
across the basolateral membrane by ABCC4. Similar results were obtained by
measurements of the transcellular transport of [14C]cytidine (data not shown). Taken
together, these results demonstrate that the highest rate of transcellular transport was
observed when recombinant ABCC4 was expressed in the basolateral membrane. In
summary we have described the double-transfected MDCK-CNT3/ABCC4 cells as a model
for apical to basolateral vectorial transport of nucleosides and nucleoside analogs.
Detection of hCNT3 and ABCC4 in human pancreas and pancreatic carcinoma. To
explore the relevance of our vectorial transport model for cancer therapy we studied the
expression pattern of hCNT3 and ABCC4 in pancreatic tissues. Pancreatic tissue (3 different
normal pancreatic samples and 3 samples from pancreatic carcinomas) were selected for
this analysis because nucleoside analogs are widely used in the chemotherapy of pancreatic
carcinoma. hCNT3 and ABCC4 expressions were analyzed in homogenates and
cryosections from surgical specimens by immunoblot analysis and immunofluorescence
microscopy using the antisera directed against hCNT3 and ABCC4 (Fig. 5). Samples from
normal kidney and renal clear cell carcinoma served as positive controls for both proteins.
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hCNT3 and ABCC4 were detected in normal pancreatic tissue and in pancreatic carcinoma,
and their relative abundance varied strongly among the different tissue samples (Fig. 5A).
The antiserum against hCNT3 identified two major bands at 90 to 100 kDa suggesting a
different extent of glycosylation of the protein in the tissues. ABCC4 was detected as a broad
band at 170 kDa. The hCNT3- and ABCC4-specific signals were abolished in all samples
when the antisera against hCNT3 or ABCC4 were preincubated with the corresponding
synthetic peptides before immunoblot analysis (data not shown).
In addition to immunoblot analysis, immunolocalization of both transporters was
performed by immunofluorescence microscopy (Fig. 5B-G). hCNT3 was localized in the
basolateral and in the apical membrane of the epithelia of ducts in normal pancreas and in
ductal pancreatic carcinoma (Fig. 5B-D). However, ABCC4 was predominantly localized only
in the basolateral membrane of the ductular epithelia (Fig. 5E-G), which is in accordance with
a recently published study by König et al. (König et al., 2005). In addition, hCNT3 and
ABCC4 were also localized in the plasma membrane of acinar cells. Na+/K+-ATPase, a
marker for the basolateral plasma membrane domain of epithelial cells, clearly colocalized
with the basolateral staining of hCNT3 and ABCC4 in the ductular epithelia (Fig. 5B-G).
Preincubation of the antibodies with the corresponding synthetic peptides abolished the
staining in the tissue cryosections (data not shown). Thus, these results demonstrate the co-
expression of hCNT3 and ABCC4 in the same cell types from normal pancreas and
pancreatic carcinoma.
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Discussion
Up until now, transport of nucleosides and nucleoside analogs across the plasma
membrane was predominantly analyzed by using single-transfected cells expressing either
an uptake transporter or an efflux pump. However, the intracellular nucleoside concentrations
are controlled both by the uptake of the compounds into the cells and by the efflux of their
metabolites into the extracellular space. Thus, the development of cell lines stably
expressing both an uptake transporter and an efflux pump for nucleosides, nucleoside
analogs and their metabolites, allows the integrated analysis of uptake and efflux. In
polarized MDCK cells, expression of recombinant uptake and efflux transporters that are
sorted into distinct membrane domains allows vectorial transport, which comprises the
uptake of compounds, their metabolism, and the efflux. These cell lines reflect the in vivo
state found in specialized epithelial tissues, but also in non-epithelial tissues (Keppler, 2005).
For this aim, we established a stable single-transfected MDCK cell line expressing
recombinant hCNT3 and a stable double-transfected MDCK cell line expressing recombinant
hCNT3 together with ABCC4. Studies on expression and localization of the recombinant
proteins by immunoblot and immunofluorescence microscopy confirmed that hCNT3 and
hABCC4 are expressed in the apical and basolateral plasma membrane of the double-
transfected MDCK cells, respectively (Fig. 1C-N). Thus, the distinct localization of each
transporter in different cell membrane domains of our cell system allow to measure vectorial
transport. This scenario reflects the in vivo state in some epithelial cells, and the differential
membrane domain localization is also essential for the technically accurate transport
measurements. Nevertheless, several non-epithelial tumor cells treated with nucleoside
analogs showed co-expression of both transporters hCNT3 and ABCC4, or at least one of
them, including leukemia cells (Peng et al., 2008; Schuetz et al., 1999; Molina-Arcas et al.,
2003; Fotoohi et al., 2006; Guo et al., 2009) and solid tumor (Marechal et al., 2009; Norris et
al., 2005; Fig. 5).
The functionality of hCNT3 was examined in our MDCK-CNT3 transfectants. The
MDCK-CNT3 cells were able to transport the known substrates cytidine, uridine, and
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5-fluoro-5’-deoxycytidine (Fig. 2B-C and Table 1; Ritzel et al., 2001). The kinetic analysis of
these substrates revealed Km values in the micromolar range (Table 1), similar as described
previously (Ritzel et al., 2001). Recently, we identified hCNT1 as a novel nucleoside
transporter for the anticancer drug 5-azaCyd (Rius et al., 2009), a nucleoside analog, the
transport of which had only been studied in cell systems with endogenous transporters
(Huang et al., 2004; Rius et al., 2009). Our present work demonstrated that hCNT3 also
mediates 5-azaCyd uptake into the cells with a Km value of 147 µM (Fig. 2A and Table 1) and
suggests that hCNT3 also mediates uptake of the structurally related drug 5-aza-2'-
deoxycytidine (Table 2). In agreement with this notion, the expression of hCNT3 also
resulted in a higher sensitivity of the cells to 5-azaCyd and 5-aza-2'-deoxycytidine (Table 2).
This finding is in line with the relatively broad substrate specificity of hCNT3. Since 5-azaCyd
is an emerging epigenetic drug that has found increasing use for the treatment of myeloid
leukemias and solid tumors (Silverman and Mufti, 2005; Appleton et al., 2007; Stresemann
and Lyko, 2008), it will be interesting to determine whether the expression level of hCNT3 is
relevant in the clinical response to 5-azaCyd.
The double-transfected polarized MDCK-CNT3/ABCC4 cells show an opposite
localization as the transport proteins required for vectorial transport described previously e.g.
in the OATP1B3/ABCC2 double-transfected cells (Cui et al., 2001). In this work here, the
uptake transporter hCNT3 was localized to the apical membrane of MDCK cells, while the
export pump ABCC4 is localized to the basolateral membrane of MDCK cells (Fig. 1).
Therefore, vectorial transport across these cells could be significant only from the apical to
the basolateral compartment, since both transporters mediate unidirectional transport (Fig.
3A and 4A). Indeed, vectorial transport of cytidine and 5-azaCyd by the MDCK-CNT3 and
MDCK-CNT3/ABCC4 was detected significantly only from the apical to the basolateral
membrane and in a Na+-dependent manner, while transport from the basolateral to the apical
chamber was insignificant (Fig. 3B-C and Fig. 4B-D). As such, we describe here for the first
time an in vitro cell model for measurements of the vectorial transport of nucleosides and
nucleoside analogs by unidirectional transport proteins from the apical to the basolateral
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membrane. A recent study reported that a single-transfected MDCK cell line expressing
recombinant hCNT3 in the apical membrane showed increased apical-to-basolateral
transport of cytidine (Errasti-Murugarren et al., 2007). However, the efflux transporters in the
basolateral membrane of the cells were of endogenous origin. In contrast, our double-
transfected cells express recombinant ABCC4 as an active ATP-dependent efflux pump, able
to transport substrates with high efficiency. Further studies are necessary to elucidate
whether reduced glutathione might play a role in the efflux of derivatives of nucleosides and
nucleoside analogs by ABCC4, as previously reported for other substrates (Lai and Tan,
2002) and especially for bile acids (Rius et al., 2003; Rius et al., 2008).
As expected, 5-azaCyd was significantly accumulated only in a sodium-dependent
manner in the MDCK-CNT3 and MDCK-CNT3/ABCC4 cells and was detected only in the
basolateral compartment of the MDCK-CNT3 and MDCK-CNT3/ABCC4 cells (Fig. 4B-D).
The detection of compounds in the basolateral compartment of MDCK-CNT3 cells indicated
the presence of endogenous equilibrative or efflux transporters in the cells. MDCK cells are
known to express a variety of endogenous transporters and some of them have been
identified as equilibrative uptake transporters, such as Ent1 (Hammond et al., 2004) and as
the canine efflux pump Abcc4 (Bartholomé et al., 2007). NBTI, which is an inhibitor of hENT1
at nanomolar concentrations and a micromolar inhibitor of hENT2 (Zhang et al., 2007),
strongly increased the sodium-dependent intracellular accumulation of 5-azaCyd and cytidine
in the MDCK-CNT3 and MDCK-CNT3/ABCC4 as compared to MDCK-Co in the presence or
absence of NBTI (Fig. 4). In contrast, the basolateral amount of 5-azaCyd and cytidine in the
presence of NBTI was reduced compared to the absence of NBTI (Fig. 3C). Together, these
data establish our MDCK-CNT3/ABCC4 cells as a model for vectorial transport from the
apical to the basolateral membrane. In addition, the endogenous expression of the canine
Ents in the MDCK cells reflects the ubiquitous expression of ENTs under physiological
conditions. Thus, the apical-to-basolateral transport system may provide new insights into
cell types that are clinical relevant. Importantly, coexpression of hCNT3 and ABCC4 could be
demonstrated in human pancreas and pancreatic carcinoma. The localization of hCNT3 in
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both plasma membrane domains of the pancreatic epithelial cells suggests a key role of the
transporter in the basolateral membrane by taking up nucleoside-derived drugs from the
blood side, while its expression in the apical membrane served a physiological function of
uptake and salvage of nucleosides present on the luminal side of the epithelial ducts.
Nucleoside analogs play an important role in the clinical management of pancreatic cancer
and it will be of interest to investigate the roles of hCNT3 and ABCC4 in therapy responses.
It is of interest in this context that recent studies in patients with resected pancreatic
adenocarcinoma undergoing chemotherapy with gemcitabine described hCNT3 as a marker
for longer survival (Marechal et al., 2009).
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Footnotes
*This work was supported by the German Cancer Research Center, Heidelberg, Germany;
the Wilhelm Sander Foundation, München, Germany (Grant 2004.101.1) and the Deutsche
Krebshilfe, Bonn, Germany (grant 107150). Maria Rius was supported by the Peter und
Traudl Engelhorn-Stiftung zur Förderung der Biotechnologie und Gentechnik.
Address for reprints: Maria Rius, Division of Epigenetics, German Cancer Research Center,
Im Neuenheimer Feld 580, 69120 Heidelberg, Germany.
Phone: +49-6221-423806
Fax: +49-6221-423802
E-Mail: [email protected]
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Legends for figures
Fig. 1. Expression and immunolocalization of recombinant hCNT3 and ABCC4 in MDCK
cells. Immunoblot analysis of hCNT3 (A) and ABCC4 (B) in crude membrane fractions
prepared from MDCK cells stably transfected with control vector (Control), with hCNT3 cDNA
(CNT3), or with hCNT3 and ABCC4 cDNAs (CNT3/ABCC4) (10 µg of protein per lane). The
blots were immunostained using the antisera against hCNT3 (A) or ABCC4 (B). (C-N) Cells
were grown in a polarized fashion on membrane inserts and analyzed by confocal laser
scanning microscopy for the localization of hCNT3 (red; C-H) and of ABCC4 (green; I-N).
Nuclei are stained in blue; D, F, H, J, L, and N are vertical sections through the cell
monolayers at positions indicated by the white lines in C, E, G, I, K, and N. Scale bars, 10
µm.
Fig. 2. Transport of [14C]Cytidine, [3H]uridine, [3H]5-fluoro-5'-deoxyuridine (FdUrd), and
[14C]5-azacytidine (5-azaCyd) by hCNT3. MDCK control cells (Control) and hCNT3-
expressing cells (CNT3) were grown as described under Methods. Cells were incubated with
1 µM [14C]cytidine (A), 1 µM [3H]uridine (B), 1 µM [3H]FdUrd (C), or 1 µM [14C]5-azaCyd (D) in
the presence of 100 mM NaCl (closed symbols) or in the presence of 100 mM choline
chloride (open symbols). Intracellular substrate accumulation is presented as means ± SD,
determined from a triplicate determination reproduced independently twice.
Fig. 3. Vectorial transport in MDCK cells expressing hCNT3. A, schematic outline of single-
transfected MDCK cells expressing the human apical nucleoside uptake transporter hCNT3.
Polarized cells were grown on a filter membrane support for studies on vectorial transport. B,
vectorial transport of [14C]cytidine. The MDCK control cells (MDCK-Co) and the hCNT3-
expressing cells (MDCK-CNT3) were grown on cell culture inserts as described under
Methods. [14C]Cytidine (1 µM) was given either to the apical compartment (A) or to the
basolateral compartment (B). After 15 min at 37°C, radioactivity in the opposite
compartments was determined to calculate the transcellular transport. C, inhibition of the
[14C]cytidine vectorial transport by NBTI. [14C]Cytidine (1 µM) was given to the apical
compartment and NBTI (10 µM) to the basolateral compartment. After 15 min at 37°C,
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radioactivity in the basolateral compartment was measured. Transcellular transport in the first
15 min was in the linear range. Data represent means ± SD determined from a triplicate
determination.
Fig. 4. Vectorial transport in MDCK cells expressing hCNT3 and ABCC4. A, schematic
outline of double-transfected MDCK cells expressing the human apical nucleoside uptake
transporter hCNT3 together with the human efflux pump ABCC4. Polarized cells were grown
on a filter membrane support for studies on vectorial transport of substances that are
substrates for the hCNT3 as well as for ABCC4. B-D, Vectorial transport of [14C]5-azaCyd.
MDCK control cells (MDCK-Co, Control) and the stably transfected cells (MDCK-CNT3 and
MDCK-CNT3/ABCC4) were grown as described under Methods, and incubated with 1 µM (B-
C) or 10 µM (D) [14C]5-azaCyd in the apical chamber. NBTI (10 µM) was added to the
basolateral compartment (C) as indicated. Experiments were performed in the presence of
100 mM NaCl or in the presence of 100 mM choline chloride. After incubation for 15 min,
radioactivity in the basolateral chamber was determined (lower panel). Intracellular substrate
accumulation was calculated after lysing the cells (upper panel). Transcellular transport in
the first 15 min was in the linear range. *, p < 0.05, compared with values obtained from
MDCK-CNT3 cells in Na+-containing buffer. Data represent means ± SD from a triplicate
determination reproduced independently twice.
Fig. 5. Expression and immunolocalization of hCNT3 and ABCC4 in human normal and
malignant pancreatic tissues. A, immunoblot analysis of hCNT3 and ABCC4 in homogenates
(50 µg of total protein per lane) from normal pancreatic tissue and in pancreatic carcinoma
and from human kidney, as a positive control. hCNT3 was detected with the NTS antiserum
(anti-CNT3) as 3 major bands at 90 to 100 kDa and ABCC4 was detected with the SNG
antiserum (anti-ABCC4) as a broad band at 170 kDa. B-G, Confocal laser scanning
micrographs of cryosections from normal human pancreatic tissue (B-C and E-F) and from
human pancreatic ductal adenocarcinoma (D and G) stained with the NTS antiserum (B-D,
green) and with the affinity-purified SNG antibody (E-G, green). The Na+/K+-ATPase (B-G,
red) was used as a marker for the plasma membrane and the basolateral membrane domain
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of epithelial cells. B to D, localization of hCNT3 in the apical (a) and basolateral (b)
membrane of the ductular epithelial cells (arrow) and in the plasma membrane of acinar cells
(arrowhead). E to G, localization of ABCC4 in the basolateral membrane of the ductular
epithelial cells (arrow) and in acinar cells (arrowhead). Colocalization of hCNT3 or ABCC4
(green) with the Na+/K+-ATPase (red) confirms the basolateral localization of these proteins
(merged yellow color). Nuclei were stained with DAPI (B-G, blue). Scale bars, 20 µm.
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Table 1. Kinetic analysis for hCNT3-mediated uptake of labeled nucleosides.
Substrate Km Vmax Vmax/Km
µM nmol x mg protein-1 x min-1 µl x mg protein-1 x min-1
5-AzaCyd 147 ± 17 43 ± 6 294
Cytidine 151 ± 15 30 ± 3 196
Uridine 51 ± 10 8 ± 1 147
5-F-5'-dUrd 615 ± 41 59 ± 18 96
Rates of Na+-dependent intracellular accumulation of [14C]5-azaCyd, [14C]cytidine,
[3H]uridine, and of [3H]5-F-5'-dUrd were determined in hCNT3-transfected MDCK cells under
substrate concentrations described under Methods. The Km values were calculated from
double-reciprocal plots. Data represent mean values ± S.D. from a triplicate determination.
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Table 2. Effect of 5-azaCyd and 5-aza-2'-deoxycytidine on the sensitivity of
hCNT3-expressing MDCK cells.
IC50* (µM)
Nucleoside analog MDCK-Co MDCK-CNT3 Relative
sensitivity**
5-AzaCyd 54 ± 5 0.8 ± 0.07 67.5
5-Aza-2'-dCyd 331 ± 48 25 ± 4 13.2
MDCK-Co and MDCK-CNT3 cells were incubated with different concentrations of 5-azaCyd
and 5-aza-2'-dCyd for 72 h, and the cell viability was determined by AlamarBlue assays (n =
8).
*IC50 is the nucleoside analog concentration required to reduce cell survival by 50 %.
**The relative sensitivity is the IC50 for nucleoside analog treatment in MDCK-Co cells divided
by the IC50 in MDCK-CNT3 cells.
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ABCC4Nuclei
CNT3 ControlCNT3/ABCC4
ABCC4Nuclei
ABCC4Nuclei
Control
CNT3Nuclei
CNT3/ABCC4
CNT3Nuclei
CNT3Nuclei
CNT3
A B
C GE
I MK
Figure 1
Dxz
Fxz
Hxz
Jxz
Lxz
Nxz
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Tra
nsc
ellu
lar
[14 C
]Cyt
idin
e T
ran
spo
rt in
toB
aso
late
ral C
ham
ber
(nm
ol/m
g p
rote
in a
t 15
min
)
0.0
0.2
0.4
0.6
- NBTI + NBTI
Control
CNT3
Control
CNT3
NaClChoCl
Basolateral
Tight Junction
Apical
hCNT3
Filter membrane
Figure 3
A
C
0.0
0.2
0.4
0.6
ABA B NaClChoCl
Tra
nsc
ellu
lar
[14 C
]Cyt
idin
e T
ran
spo
rt
(nm
ol/m
g p
rote
in a
t 15
min
)
Control
CNT3
Control
CNT3
B Apical Basol. Basol. Apical
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Intr
acel
lula
r[14
C]5
-Aza
Cyd
Acc
um
ula
tio
n(n
mo
l/mg
pro
tein
at 1
5 m
in)
0.0
0.3
0.6
0.9
CNT3/A
BCC4
CNT3/ABCC4
Control
CNT3
Control
CNT3
Tra
nsc
ellu
lar
[14C
]5-A
zaC
yd T
ran
spo
rt in
toB
aso
late
ral C
ham
ber
(nm
ol/m
g p
rote
in a
t 15
min
)
0.0
0.4
0.8
1.2
0.0
0.1
0.2
NaClChoCl
1 µM 5-azaCyd- NBTI
0.0
0.3
0.6
0.9
1 µM 5-azaCyd+ 10 µM NBTI
0
1
2
3
4
0
1
2
3
CNT3/ABCC4
Control
CNT3
10 µM 5-azaCyd- NBTI
ABCC4
hCNT3
Filter membrane Basolateral
Tight Junction
Apical
A
Figure 4
B C D
*
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Nuclei hCNT3 Na+/K+-ATPase Merge
B
C
D
E
F
G
A
Figure 5
Nuclei ABCC4 Na+/K+-ATPase Merge
b
a
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